Laser Time-Resolved Fluorescence Studies for Investigating Chromophore-Biosubstrate Interactions

Abstract

Fluorimetric studies utilizing continuous wave (cw) techniques performed on chromophores that bind specifically to the biomacromolecule or to the cell organelle or compartment to be examined allowed a great deal of information to be gained, in the past, on biomolecular structures and kinetics (Udenfried, 1969). Flow cytometry, which has become such an important methodology for auto mated cytology, is just an example of the extent to which the fluorescence properties of Acridines, ie. dyes exhibiting selective affinity for nucleic acids (Albert, 1966), have been exploited to determine, for instance, DNA and RNA contents of single cells or chromosomes aberrations or cell-cycle phases (Mullaney et al., 1974). These investigations basically take advantage of the fact that the optical properties - fluorescence, in particular - of chromophores are affected unequivocally by binding to biomacromolecules or biostruc- tures (Andreoni, 1985). Moreover, the stronger and/or more disruptive is the interaction with the substrate, the more pronounced is, usually, the effect of the binding on the chromophore emission. This property is so general that variations observed both “in vitro” and “in vivo” in the fluorescence quantum yield or in the emission spectrum of fluorescent drugs are commonly considered to be indicative of the stability of their complexation (Geacintov, 1987) or of their binding to specific cell substances or sites (Goormaghtigh et al., 1980; Kessel et al., 1985)